The widespread need to inactivate a protein in its cellular context is reflected in the degree to which the research community has embraced RNAi technology. But RNAi constructs, even when effective, can be slow to take effect as they depend on the turnover of pre-existing protein. As the protein levels in the cell decline, the cell may die or compensatory mechanisms may be activated. In many instances, it would be desirable to rapidly inactivate a protein with minimal side effects on other cellular components.
Light-triggered methods have been developed in several labs. Examples of light-based methods for protein inactivation include CALI and FAL1 (Chromophore or Fluorophore-Assisted Laser Inactivation) in which an illuminated fluorophore produces a local shower of reactive oxygen species to damage the protein to which it is attached (1). However, the method may not be fully effective and may cause collateral damage to proteins in close proximity (2). Therefore other methods are in development including light-dependent uncaging of inhibitory drugs and methods that harness the light-induced conformational changes of LOV domains to regulate a coupled protein or peptide (3). However, of the available approaches, no single method will meet all needs Inhibitors are not available for many cellular proteins and not all proteins will be susceptible to photoregulation when coupled to an LOV domain. Another approach has been to convey pharmacological sensitivity on a protein by coupling it to a peptide sequence or domain that is sensitive. Introduction of the target sequence for Hepatitis C Virus NS3 Protease, for example, may sensitize a protein to expression of this protease; which can itself be regulated by inhibitory drugs (4).
Proteins may be coupled to binding sites for high-affinity ligands that will cause them to be dimerized or selectively localized to a designated subcellular compartment. Many Chemical Inducers of Dimerization (CID) have been developed and used to cross-link or localize proteins in the cell. These CIDs include FK1012 (5, 6), rapamycin (6), AP21967 (7) and iRAP (8) and can induce either homodimerization or heterodimerization and thereby activate or inactivate certain proteins or promote their degradation. CIDs have been adopted for a broad array of cellular components because they can be applied to any protein that can tolerate the addition of the necessary binding sites. However, not all proteins can be controlled in this fashion; success depends on whether the dimerization successfully alters the function of the protein. The time course of their action depends on the rate at which they will enter the cell and reversal of the drug requires slow washout and/or competition with a monovalent ligand.
There is a need in the art for an agent that allows for modulation of protein activity with minimal side effects on other cellular components.